Oral cyclosphorin formulations

ABSTRACT

Improved oral cyclosporin formulations which have high bioavailability and are capable of admiration in hard capsules are provided. In the subject formations, cyclosporin is delivered in an orally acceptable vehicle comprising at least one alkanol solvent of from 2 to 3 carbon atoms in combination with at least one non-ionic surfactant. The subject formalations may further comprise at least one cosolvent, where cosolvents of interest include fatty acids and diols. The subject formulations find use in immuno-suppressive therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/620,021 filed Mar. 21,1996, now U.S. Pat. No. 5,766,629 which is a now U.S. Pat. No.5,834,017, continuation-in-part of application Ser. No. 08/519,689 filedAug. 25, 1995, which application is herein incorporated by reference.

INTRODUCTION

1. Field of the Invention

The field of this invention is oral cyclosporin formulations.

2. Background

Despite efforts to avoid graft rejection through host-donor tissue typematching, in the majority of transplantation procedures where a donororgan is introduced into a host, immunosuppressive therapy is criticalto the maintained viability of the donor organ in the host. A variety ofimmunosuppressive agents have been employed in transplantationprocedures, including azathioprine, methotrexate, cyclophosphamide,FK-506, rapamycin and corticosteroids. Agents finding increased use inimmunosuppressive therapy due to their preferential effect on T-cellmediated reactions are the cyclosporins.

Cyclosporins are a class of cyclic polypeptides consisting of elevenamino acids which are produced as a metabolite by the fungus speciesTolypocladium inflatum Gams. Cyclosporins have been observed toreversibly inhibit immunocompetent lymphocytes, particularlyT-lymphocytes, in the G₀ or G₁ phase of the cell cycle. Cyclosporinshave also been observed to reversibly inhibit lymphokine production andrelease. Although a number of cyclosporins are known, Cyclosporin A isthe most widely used.

Use of Cyclosporin A has been reported to prolong the survival ofallogeneic transplants involving skin, heart, kidney, pancreas, bonemarrow, small intestine and lung. In allogeneic transplantations,Cyclosporin A has been shown to suppress humoral immunity and, to agreater extent, cell mediated immune reactions, including: allograftrejection, delayed hypersensitivity, experimental allergicencephalomyelitis, Freund's adjuvant arthritis, and graft vs. hostdisease. Although success has been realized with Cyclosporin A,following transplantation administration of the agent must be continuedsince the benefits of cyclosporin therapy are reversible and graftrejection occurs once administration of Cyclosporin A is discontinued.

Although cyclosporin formulations for both oral and intravenousadministration have been developed, oral administration of cyclosporinis preferred because of the ease of administration and greater patientacceptance. Furthermore, intravenous administration of cyclosporin canresult in anaphylactic reactions, a side effect not observed with oralformulations. Oral cyclosporin formulations which have been developedand are currently marketed include both soft gelatin capsule andsolution formulations, both of which are sold under the trademarksSANDIMMUNE® and NEORALS™.

In using oral cyclosporin formulations in immunosuppressive therapy,both the care giver and manufacturer must be cognizant of many issues.With oral cyclosporin formulations, cydosporin bioavailablity can belimited because of cyclosporin's immiscibility in water and the tendencyof cyclosporin to precipitate in aqueous environments. In addition, theconcentration of cyclosporin present in oral formulations can be limiteddue to cyclosporin's hydrophobic nature. Furthermore, cyclosporinabsorption by the gastrointestinal tract can be erratic from oneformulation batch to the next, requiring constant monitoring ofcyclosporin blood levels during treatment. Finally, packaging andstorage stability are an issue with oral formulations. For example, withsoft gelatin capsule formulations of cyclosporin, air tight packagingmust be employed, which is inconvenient due to bulkiness and high cost.Furthermore, cyclosporine formulations may be unstable at lowertemperatures, as cyclosporine crystalization may occur.

Thus, desirable oral cyclosporin formulations would be formulations thataddress at least some of the above issues. Ideally, oral formulationswould promote high bioavailability, comprise high concentrations ofcyclosporin and would be amenable to preparation in hard capsule form.

Relevant Literature

Physician's Desk Reference (1994) pp 2071-2074 describes oralcyclosporin formulations curly sold under the trademark SANDIMMUNE®.

Oral cyclosporine formulations are also described in the NEORAL™ packageinsert, (1995) (Sandoz Pharmaceuticals Corporation, East Hanover, N.J.,07936).

U.S. Patents of interest describing cyclosporins and derivatives thereofinclude: 4,220,641; 4,639,434; 4,289,851; and 4,384,996. U.S. Pat. No.5,047,396 describes an intravenous preparation for administration ofcyclosporin. U.S. Pat. Nos. 4,388,307; 4,970,076 and 4,990,337 describethe preparation of oral cyclosporin formulations.

The preparation of hard capsules for the oral delivery of pharmaceuticalformulations is described in U.S. Pat. Nos. 4,822,618; 4,576,284;5,120,710; and 4,894,235.

BRIEF SUMMARY OF THE INVENTION

Oral cyclosporin formulations, and methods for their use inimmunosuppressive therapy, are provided. In the subject formulations,cyclosporin is present in an orally acceptable vehicle comprising atleast one alkanol solvent of from 2 to 3 carbon atoms in combinationwith at least one non-ionic surfactant. The subject formulations mayfurther comprise one or more cosolvents, where cosolvents of interestare fatty acid esters and diols. The cyclosporin formulations can bepackaged as hard capsules.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the cyclosporin peak concentration (C_(max)) achieved inrats for several oral formulations according to the subject invention,where the C_(max) is shown as a relative value compared to the C_(max)achieved with SANDIMMUNE® ORAL formulation (SO).

FIG. 2 provides the time at which C_(max) occurred (T_(max)) for each offormulations shown in FIG. 1, where T_(max) is provided as relativevalue compared to the T_(max) of SANDIMMUNE® ORAL formation (SO).

FIG. 3 provides the relative area under the blood concentration-timecurve (AUC) for each of the formulations shown in FIG. 1, where AUC isprovided as a relative value compared to the AUC value for SANDIMMUNE®ORAL formulation (SO).

FIG. 4 provides the cyclosporin peak concentration (C_(max)) achieved inhumans for several oral formulations according to the subject invention,as well as SANDIMMUNE® ORAL solution (“Sand” in the figure).

FIG. 5 provides the time at which C_(max) occurred (T_(max)) for each offormulations shown in FIG. 4.

FIG. 6 provides the area under the blood concentration-time curve (AUC)for each of the formulations shown in FIG. 4.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Oral cyclosporin formulations are provided which promote bioavailabilityand can be formulated as capsules, particularly hard capsules. In thesubject formulations, cyclosporin is present in an orally acceptablevehicle comprising at least one alkanol solvent of from 2 to 3 carbonatoms in combination with at least one non-ionic surfactant. The subjectformulations may father comprise at least one cosolvent, wherecosolvents of interest include fatty acid esters and diols. Each of thecomponents of the subject formulations are pharmaceutically acceptable.In addition to providing for high bioavailability, the subjectformulations provide for reproducible cyclosporin absorption from onebatch of a particular formulation to the next. The subject formulationsfind use in immunosuppressive therapy.

A number of cyclosporins are known in the art to exhibitimmunosuppressive activity and may be delivered in the subject oralformulations. Cyclosporins that may be administered in the subjectformulations include Cyclosporin A, Cyclosporin B, Cyclosporin C,Cyclosporin D and Cyclosporin G, as well as synthetic analogs thereofSee Merck Index (1989) 2759. The subject oral formulations areparticularly suited for the delivery of Cyclosporin A. When delivered inthe subject formulations, Cyclosporin A will be present inconcentrations ranging from 50 to 150 mg/ml, usually 100 to 150 mg/ml,based on the volume of the vehicle component of the formulation.

The vehicle component of the subject formulations will include analkanol solvent component, where the alkanol solvent component willcomprise at least one alkanol and usual no more than three differentalkanols, more usually no more than two different alkanols, where thealkanols will usual be from 2 to 3 carbon atoms, and from 1 to 2 hydroxygroups, such that there is no more than 1 hydroxy group per 1.5 carbonatoms. Suitable alkanols include ethanol and propylene glycol. The totalamount of alkanol solvent in the formulation will be at least about 1%(v/v), usually at least about 3% (v/v) and may be as high as 95% (v/v),but will generally range from about 5 to 75% (v/v), by from about 5 to60% (v/v), and more usually from about 10 to 60% (v/v) of theformulation. When ethanol is present in the formulation as an alkanolsolvent, the amount of ethanol may range from 5 to 20% (v/v), usuallyfrom about 5 to 15% (v/v) of the formulation, while when propyleneglycol is present as an alkanol solvent, the amount of propylene glycolin the subject formulation may range from about 5 to 90% (v/v), usuallyfrom about 5 to 85% (v/v), more usually from about 10 to 50% (v/v) ofthe formulation.

Also present in the orally acceptable vehicle will be at least onenon-ionic polyoxyalkylene surfactant, usually not more than twopolyoxyalkylene non-ionic surfactants. The polyoxyalkylene surfactantswill have a hydrophilic-lipophilic-balance (HLB) of from about 5 to 20,usually from about 8 to 16. Preferably, the polyoxyalkylene non-ionicsurfactants employed in the subject formulations will be polyoxyethylenecompounds. Polyoxyethylene compounds of interest include: ethoxylatedalcohols, i.e. polyoxyethylene alcohols or ethoxylated fatty alcohols,where the alcohol moieties are generally of from 10 to 18, usually from10 to 14 carbon atoms, as well as ether and ester substituents thereofand polyoxyethylene derivatives of fatty acid partial esters, usuallymonoesters, of polyols of from 4 to 6 carbon atoms, usually 6 carbonatoms, where the polyols may be polyol anhydrides e.g. sorbitan. Thefatty acid moieties of the subject surfactant will typically range from10 to 18 carbon atoms. The number of ethylenoxide groups will generallybe in the range of 2 to 30, usually in the range from about 2 to 25.Preferred surfactants are polyoxyethylene (4) lauryl ether (BRIJ 30®)and polyoxyethylene (20) mono sorbitan mono-oleate (TWEEN 80®. The totalamount of non-ionic surfactants present in the subject formulations willrange from 5 to 65%, usually from about 5 to 60% (v/v) of theformulation. Where TWEEN 80® is present in the formulation, it willusually be present in amounts ranging from 5 to 60%, more usually fromabout 10 to 50% (v/v) of the formulation. When BRIJ 30® is present inthe subject formulation, it will usually be present in amounts rangingfrom 10 to 45%, more usual from about 15 to 40% (v/v) of theformulation.

The subject formulations may further comprise one or more cosolvents,usually not more than three different cosolvents, more usually not morethan two different cosolvents, where suitable cosolvents include fattyacid esters and diols, where the cosolvent may be 100% fatty acid ester,100% diol, or combination thereof. The total amount of cosolvent presentin the formulation may range from about 20 to 80% (v/v) and will usuallyrange from about 25 to 75% (v/v). When present in the formulation, theratio of cosolvent to solvent in the subject formulations may range fromabout 1:1 to 15:1, but will usually range from about 1:1 to 13:1.

Fatty acid esters which may serve as cosolvents in the subjectformulations are those fatty acid esters where the hydrocarbon chain ofthe fatty acid is from 12 to 18, usually 14 to 18 carbon atoms inlength, where the fatty acid ester will be a mono-ester of a loweralkanol. Suitable fatty acid esters will generally comprise an evennumbered fatty acid chain, where the hydrocarbon chain may be saturatedor unsaturated, usually having not more than two sites of unsaturation.Fatty acids of interest will generally be of plant or mammalian originand include palmitate, stearate, palmitoleate, linoleate, linolenate andthe like, particularly myristate and oleate. The alcohol of the fattyacid mono-ester will be a lower alkanol of from 2 to 4 carbon atoms inlength, usually 2 to 3 carbon atoms in length, with or without branches.Fatty acid esters of particular interest are isopropyl myristate andethyl oleate. Isopropyl myristate, when present, will range from about55 to 75% (v/v), and ethyl oleate, when present, will range from about35 to 75% (v/v) ofthe total formulation. Usually the fatty acid esterwill be present in an amount at least about equal (v/v) and up to 8times the amount of surfactant in the formulation, usually not greaterthan 5 times the amount of surfactant in the formulation (v/v).

Diols may also be present in the subject formulations, where the diolsmay be present in addition to, or in lieu of, the fatty acid estercosolvent. Diols of interest as cosolvents are generally liquids atphysiologic temperatures and include diols of from 8 to 28 carbon atoms,usually 16 to 20 carbon atoms, where the diol may be a polyoxyalkylenediol, where alkylene is of from 2 to 3 carbon atoms. Suitable diols foruse as cosolvents may range from about 200 to 800 daltons, usually fromabout 200 to 650 daltons. Diols of particular interest includepolyethylene glycols, particularly polyethylene glycol 200 (PEG₂₀₀),polyethylene glycol 400 (PEG₄₀₀), polyethylene glycol 600 (PEG₆₀₀), andthe like, with PEG₄₀₀ being preferred. When present as cosolvents in thesubject formulations, the diols will range from about 5 to 60% (v/v),usually from 5 to 55% (v/v) of the formulation.

In the subject formulations, the cosolvents themselves may impartdesirable physical properties to the formulation, such as viscosity,stability and the like. Where desired, the formulation may furthercomprise additional agents which impart desired physical properties tothe formulation, such as thickening agents, suspending agents,solidifying agents, and the like, where such agents include acacia,carboxymethylcellulose, hydroxypropylcellulose, lecithin, methylcellulose, high molecular weight polyethylene glycols, e.g thosepolyethylene glycols with molecular weights ranging from about 1000 to6000, usually 1000 to 5000 daltons, povidone, sodium alginate,tragacanth, and the like. Also present in the subject formulations maybe a number of minor components which provide various functions, such asenzyme inhibitors, preservatives, antioxidants, antimicrobial agents,stabilizers and the like. The total amount of these thickening agentsand other additives, when present in the formulation, will normally notbe greater than 5 weight %, usually 2 weight %, more usually 1 weight %of the formulation. A number of excipients may also be present in thesubject formulations, as is known in the art.

The subject formulations are stable over a wide range of temperatures,where by stable is meant that the physical integrity of the formulationis not comprised, e.g. crystallization of the cyclosporin active agentdoes not occur. Included within the temperature range over which thesubject formulations are stable are lower temperatures, such as thoseemployed in refrigerated storage, where such lower temperaturestypically range from about 0 to 15° C., more typically from about 2 to8° C.

The subject formulations are suitable for administration in capsuleform, e.g. hard and soft capsules. Methods of producing hard capsulescomprising liquid formulations are known in the art and described inU.S. Pat. Nos. 4,822,618 and 4,576,284, the disclosures of which areherein incorporated by reference. Generally, hard capsules that find usewith the subject formulations will comprise two parts: a shell componentand a cap component. The shell and cap components fit together toproduce an enclosed cavity of defined volume sealed in a hard capsuleshell. The shell and cap components may be fabricated from a bydrophilicpolymer, such as starch or gelatin. In preparing the hard capsules, theliquid formulation will be poured into the shell component and then thecapsule will be sealed by fitting the cap component over the shellcomponent. The seal between the two components may be secured, therebypreventing leakage of the enclosed formulation from the capsule, byusing a sealant as described in EP 116744, the disclosure of which isherein incorporated by reference. To avoid degradation in the stomach,capsules comprising the subject formulations may be coated with anenteric coating which inhibits degradation of the capsule in the acidicenvironment of the stomach. A variety of enteric coatings are known inthe art. See for example, U.S. Pat. No. 5,206,219, the disclosure ofwhich is herein incorporated by reference.

The subject formulations find use in immunosuppressive therapy.Immunosuppressive therapy is indicated in a wide variety of diseases,including idiopathic nephrotic syndrome, type I insulin-dependentdiabetes, Behcet's syndrome, active Crohn's disease, a plastic anemia,severe corticosteroid-dependent asthma, psoriasis, rheumatoid arthritis,and other diseases where the immune system may play a pathogenic role.Of particular interest is the use of the subject formulations intransplant situations, including both allogeneic and xenogeneic organ,tissue or cell transplantation, where immunosuppression is desired toensure maintained viability of the transplanted organ or tissue or cellfollowing transplantation, i.e. to prevent graft rejection or preventgraft vs. host disease, e.g. following bone marrow transplantation.

In using the subject formulations to provide immunosuppressive therapyto a host, an effective amount of cyclosporin will be orallyadministered to achieve the desired level of immunosuppression in thehost, depending on the particular condition to be treated. Withtransplantation, usually an initial dosage of cyclosporin will beadministered prior to operation. Following transplantation of the donororgan to the host, the cyclosporin will be administered repeatedly, i.e.chronically, to the host to maintain immunosuppression. The initialdosage will be administered 4 to 12 hours prior to transplantation andmay range from 10 to 18 mg/kg host, usually 10 to 15 mg/kg host.Following the operation, the initial dosage will usually be continued ona daily basis for a period of 1 to 3 weeks, usually 1 to 2 weeks. Thedosage may then be tapered to a maintenance dosage of 3 to 10 mg/kg perday, usually 3 to 6 mg/kg per day. The rate at which the dosage istapered to the maintenance level may range from 3 to 8% per week andwill usually be about 5% per week. The dosage will typically be adjustedbased on trough blood levels to maintain a concentration of 150 to 250ng/ml, as measured by HPLC, RIA, ELISA or TDx assay. The subjectformulations may be administered in conjunction with additional agents,where adjunct therapy is recommended and is known in the art. Forexample, the subject formulations may be administered in conjunctionwith adrenal corticosteroids, azathioprine and the like.

Administration of the subject formulations in conjunction withtransplantation of a donor organ to a host will result in a prolongationof the viability of the donor organ in the host as a result ofsuppression of the host's immune response to the presence of the donororgan. By “prolongation of viability” is meant that the donor organremains viable in the host for a longer period of time than it wouldhave had immunosuppressive therapy not been employed in conjunction withthe transplantation. Thus, prolongation of viability includesmaintenance of viability for an indefinite period of time. A donor organis considered viable as long as it maintains functionality in the hostenvironment.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

Several oral cyclosporin formulations according to the subject inventionwere prepared. The bioavailability of cyclosporin in the preparedformulations was then observed in rats and humans.

I. Oral Cyclosporin Formulations

The following oral Cyclosporin A formulations were prepared. In eachcase, 100 mg CsA, the indicated amount of surfactant, and the indicatedamount of ethanol or propylene glycol were added to a 1.0 ml volumetricflash, and the final volume of 1.0 ml was achieved by addition of asuitable volume of fatty acid ester and/or diol.

Formulation Composition 19 EtOH 0.1 ml (10%) Tween 80 300 mg (0.278 ml)IM q.s. to 1.0 ml ((0.622 ml)(531 mg) 20 EtOH 0.05 ml (5%) Brij 30 350mg (0.368 ml) IM q.s. to 1.0 ml ((0.582 ml)(496 mg) PG 0.05 ml (5%) Brij30 350 mg (0.368 ml) IM q.s. to 1.0 ml ((0.582 ml)(496 mg) 22 EtOH 0.1ml (10%) Tween 80 300 mg (0.278 ml) EO q.s. to 1.0 ml ((0.622 ml)(541mg) 23 EtOH 0.05 ml (5%) Brij 30 350 mg (0.368 ml) EO q.s. to 1.0 ml((0.582 ml) (506 mg) 24 PG 0.05 ml (5%) Brij 30 350 mg (0.368 ml) EOq.s. to 1.0 ml ((0.582 ml) (506 mg) 33 EtOH 0.1 ml (10%) Brij 30 150 mg(0.158 ml) IM q.s. to 1.0 ml ((0.742 ml)(633 mg) 34 EtOH 0.1 ml (10%)Brij 30 150 mg (0.158 ml) EO q.s. to 1.0 ml ((0.742 ml)(646 mg) 35 EtOH0.1 ml (10%) Tween 80 500 mg (0.463 ml) PG q.s. to 1.0 ml ((0.437ml)(453 mg) 36 EtOH 0.1 ml (10%) Tween 80 300 mg (0.278 ml) PG 100 mg(0.097 ml) EO q.s. to 1.0 ml ((0.525 ml)(465 mg) 37 EtoH 0.1 ml (10%)Tween 80 300 mg (0.278 ml) PEG 400 100 mg (0.088 ml) EO q.s. to 1.0 ml((0.534 ml)(464 mg) 38 EtOH 0.1 ml (10%) Brij 30 300 mg (0.316 ml) PG100 mg (0.097 ml) EO q.s. to 1.0 ml ((0.487 ml)(424 mg) 39 EtOH 0.1 ml(10%) Brij 30 300 mg (0.316 ml) PG 200 mg (0.193 ml) EO q.s. to 1.0 ml((0.391 ml)(340 mg) 40 PG 300 mg (290 ml) Brij 30 300 mg (0.316 ml) EOq.s. to 1.0 ml ((0.394 ml)(343 mg) 41 EtOH 0.05 ml (5%) Brij 30 150 mg(0.158 ml) Tween 80 100 mg (0.093 ml) EO q.s. to 1.0 ml ((0.649 ml) (565mg) 42 PG 0.05 ml (5%) Brij 30 150 mg (0.158 ml) Tween 80 100 mg (0.093ml) EO q.s. to 1.0 ml ((0.649 ml) (565 mg) 43 EtOH 0.10 ml (10%) Tween80 400 mg (0.371 ml) PG q.s. to 1.0 ml (0.529 ml) 44 EtOH 0.10 ml (10%)Tween 80 400 mg (0.371 ml) PEG₄₀₀ q.s. to 100 ml ((0.529 ml)(601 mg) 45EtOH 0.10 ml Tween 80 300 mg (0.278 ml) PG approx.250 mg (0.243 ml)PEG₄₀₀ approx.250 mg (0.220 ml) 46 EtOH 0.10 ml (10%) Tween 80 100 mg(0.093 ml) PG q.s. to 1.0 ml (0.807 ml) 48 EtOH 0.10 ml Tween 80 200 mg(0.186 ml) PG approx.250 mg (0.243 ml) PEG₄₀₀ approx.250 mg (0.220 ml)49 EtOH 0.10 ml (10%) Tween 80 600 mg (0.558 ml) PG q.s. to 1 ml (0.342ml) 50 EtOH 0.10 ml (10%) Tween 80 300 mg (0.278 ml) PG q.s. to 1.0 ml(0.622 ml) 51 EtOH 0.10 ml (10%) Tween 80 200 mg (0.186 ml) PG q.s. to1.0 ml (0.714 ml) 52 EtOH 0.05 ml (5%) Tween 80 400 mg (0.371 ml) PGq.s. to 1.0 ml (0.579 ml) PG = Propylene Glycol; EtOH ethanol Brij 30 =polyoxyethylene (4) lauryl ether Tween 80 = polyoxyethylene (20) monosorbitan mono-oleate IM = isopropyl myristate EO = ethyl oleate

II. In vivo Bioavailability Studies for Formulations 19-24 and 33-42

The bioavailability of cyclosporin in formulations 19-24 and 33-42 wasstudied as follows. As a measure of bioavailability, the followingpharmacokinetic parameters were determined: (a) the peak bloodconcentration of cyclosporin (C_(max)); (b) time required to attainC_(max) (T_(max)); and the area under the blood concentration time-curvetime (AUC). In addition to formulations 19-24 and 33-42, thebioavailability of cyclosporin in SANDIMMUNE® Oral Solution (SO) underanalogous conditions was observed for comparison purposes. For each ofthe above formulations, CsA-naive Sprague Dawley rats weighing 250-350gm were fed pelletized standard food (Agway® 3000, Granville Mill,Greensboro, N.C.) and water ad libitum. One day prior to the experiment,silicone rubber cannulae were inserted into the right jugular and rightfemoral veins under light ether anesthesia. After overnight fast, CsAwas administered by gavage.

Following administration, 200 μl blood samples were collected from thejugular vein in 0.5 ml polypropylene microfuge tubes containing 0.3 mgof lyophilized Na EDTA and vortexed immediately for 10 sec. The samplingtimes for animals subjected to oral formalations were 0, 0.5, 1, 2, 4,8, 12, 24, 36, 48 and 72 hr after administration.

CsA, including some of its metabolites, was determined in whole blood byfluorescent polarization immunoassay (FPI)(TDx, Abbot Lab.). Briefly,150 μl of the whole blood sample were quantitatively transferred to a1.5 ml microfuge tube. Cells were lysed and dissolved with 50 μl of asurfactant-containing solubilizing reagent. Proteins were thenprecipitated out with 300 μl of acetonitrile. After centrifugation, thesupernatant was subjected to the FPI assay in a TDx Autoanalyzerfollowing the procedure recommended by Abbott Diagnostics. Since the TDxassay was originally developed for human blood, some of the recommendedprocedures were modified as follows. A series of standard solutions ofknown CsA concentration were prepared by adding a known amount of CSA torat blood treated with EDTA When the CsA concentration in a sample wasexpected to be greater than 1.0 μg/ml, the blood sample was diluted10-fold in a 0.1 M-phosphate buffer at pH 7.0. For diluted samples,another calibration curve was made using a series of standard solutionscontaining known amounts of CsA, which is volume-wise 10% in rat bloodand 90% phosphate buffer.

Descriptive pharmacokinetic parameters were obtained fromnoncompartmental analyses. The peak concentration (C_(max)) and the timeat which the peak concentration occurred (T_(max)) were estimated byinspection of the raw concentration-time profile for each rat. The areaunder the blood concentration-time curve (AUC) from time 0 through thelast data point (AUC_(0→t)) was calculated according to the lineartrapezoidal procedure. The residual area under the tail of the bloodconcentration-time curve (AUC_(t→∞)) was estimated as the ratio of thefinal observed concentration (C*) to the first-order rate constantassociated with the terminal elimination phase of the concentration-timeprofile (λ_(z)). The rate contact λ_(z) was determined by log-linearregression of the concentration-time data in the apparent terminallog-linear phase of the concentration-time profile (i.e., the final 3 to5 data points, depending on the profile under analysis). The total AUC(AUC_(t→∞)) was taken as the sum of AUC_(0→t) and AUC_(t→∞).

The results for each formation were compared with the results obtainedfor SO, and are provided in FIGS. 1-3. The results demonstrate that, forthe majority of the formulations, greater bioavailablity of cyclosporinis achieved with the subject formulations as compared with SANDIMMUNE®Oral Solution (SO), as indicated by the higher AUC values of the subjectformulations.

III. In vivo Human Bioavilability of Formations 35, 43-46 and 48-52.

48 healthy males between the ages of 19 and 55 with no more than 20%deviation from ideal weight were used as test subjects. A single dose,fasted, randomized, double-blinded, three-way crossover study wasconducted. The 48 subjects were randomized into 6 groups of 8 subjects.Each group received a single 300 mg dose of cyclosporin from the aboveformulations, or SANDIMMUNE® Oral Solution (SO), on three differentoccasions, where each occasion was separated by a 7-day washout period.

Subjects were required to fast 10 hours prior to, and 4 hours after,dosing. Water was allowed ad lib during the study, except for a 1 hourperiod prior through 2 hours following dosing. Prior to dosing, a 15 mlblood sample was drawn. For administrations, 3 ml aliquots offormulation (300 mg) was combined with 200 ml chocolate milk and orallyingested. 10 ml blood samples were drawn at t=0, 0.5, 1, 1.5, 2, 3, 4,6, 8, 10, 12, 16, 20 and 24 hours. A post study 15 ml blood sample wasalso drawn.

Concentrations of cyclosporin A in the whole blood samples were assayedusing the TDx (Abbott Diagnostics, N. Chicago, Ill.) according to themanufacturer's instructions.

Non-compartmental pharmacokinetics were derived using standard methods.The maximum whole blood concentration (C_(max)) and the time of itsoccurrence (T_(max)) were compiled from the concentration-time data. Thearea under the blood concentration time curve (AUC) was calculated bythe linear trapezoidal rule to the last blood concentration above thelimit of sensitivity (25 ng/nl) and extrapolated to infinity.

The observed C_(max), T_(max) and AUC values for each formulation wereaveraged. The average values for each formulation are provided in FIGS.4-6. The results demonstrate that for each formulation tested, Cmaxoccurred at least twice as fast as with SANDIMMUNE® Oral Solution (SO)under the same conditions. Furthermore, the AUC observed for the testformulations was at least 2000 ng·hr/ml greater than that observed forSANDIMMUNE® Oral Solution (SO) under the same conditions. Based on theseresults, formulations 35, 43-46 and 48-52 provide for greaterbioavailability than SANDIMMUE® Oral Solution (SO).

From the above results and discussion, it is evident that novelcyclosporin formulations having high bioavailability are provided. Thesubject formulations are capable of comprising high concentrations ofcyclosporin and are storage stable over a wide range of temperatures,including low temperatures commonly used in refrigeration. The subjectformulations are amenable to delivery in capsule form, including hardcapsule form, proving for ease of storage and handling.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A cyclosporin formulation consisting essentiallyof: cyclosporin; at least one alkanol solvent of from 2 to 3 carbonatoms; at least one non-ionic polyoxyalkylene surfactant, wherein saidsurfactant is selected from the group consisting of polyoxyethylenealcohols and fatty acid monoesters of ethoxylated polyols of from 4 to 6carbon atoms; and at least one cosolvent, wherein said cosolvent isselected from the group consisting of mono-esters of a lower alkanol anda fatty acid of from 14 to 18 carbon atoms and diols of from 8 to 28carbon atoms.
 2. The formulation according to claim 1, wherein saidalkanol solvent is from about 5 to 75% (v/v) of said formulation.
 3. Theformulation according to claim 1, wherein said at least one non-ionicpolyoxyalkylene surfactant is from about 5 to 65% (v/v) of saidformulation.
 4. The formulation according to claim 2, wherein said atleast one cosolvent is from about 20 to 80% (v/v) of said formulation.5. A cyclosporin formulation consisting essentially of: Cyclosporin A;at least one alkanol solvent selected from the group consisting ofethanol and propylene glycol, wherein said alkanol solvent is from about5 to 75% (v/v) of said formulation; at least one non-ionicpolyoxyethylene surfactant, wherein said non-ionic polyoxyethylenesurfactant is selected from the group consisting of polyoxyethylenealcohols and mono-esters of ethoxylated sorbitans, and is from about 5to 65% (v/v) of said formulation; and at least one cosolvent, whereinsaid at least one cosolvent is selected from the group consisting of amono-ester of a lower alkanol of from 2 to 4 carbon atoms and a fattyacid of from 14 to 18 carbon atoms, wherein said cosolvent is from about20 to 80% (v/v) of said formulation.
 6. The formulation according toclaim 5, wherein said non-ionic surfactant is selected from the groupconsisting of polyoxyethylene (4) lauryl ether and polyoxyethylene (20)mono sorbitan mono-oleate.
 7. The formulation according to claim 5,wherein said formulation comprises two cosolvents, wherein one saidcosolvents is a diol of from 8 to 28 carbon atoms.
 8. The formulationaccording to claim 7, wherein said diol is a polyoxyethylene glycol. 9.A hard capsule cyclosporin formulation consisting essentially of: a hardcapsule containing the oral formulation according to claims 1 or
 5. 10.A method for achieving immunosuppression in a host, said methodcomprising: administering to said host a cyclosporin formulationaccording to claims 1 or 5; whereby immunosuppression in said host isachieved.
 11. The method according to claim 10, wherein said at leastone alkanol solvent is selected from the group consisting of ethanol andpropylene glycol and is from about 5 to 75% (v/v) of said formulation.12. The method according to claim 10, Wherein said at least onenon-ionic polyoxyethylene surfactant is selected from the groupconsisting of polyoxyethylene (4) lauryl ether and polyoxyethylene (20)mono sorbitan mono-oleate.
 13. The method according to claim 10, whereinat least one of said at least one cosolvents is a said fatty acid esterselected from the group consisting of isopropyl myristate and ethyloleate.
 14. A method according to claim 10, wherein at least one of saidat least one cosolvents is polyethylene glycol
 400. 15. A method forprolonging the viability of a donor organ in a host, said methodcomprising; orally administering to said host in conjunction withtransplantation of said donor organ a cyclosporin formulation accordingto claim
 1. 16. The method according to claim 15, wherein saidformulation is administered in a hard capsule.
 17. The method accordingto claim 15, wherein said formulation is administered chronically aftersaid transplantation.